Role of Toll-like receptor (TLR) 2 in experimental Bacillus cereus endophthalmitis.

Bacillus cereus causes a uniquely rapid and blinding intraocular infection, endophthalmitis. B. cereus replicates in the eye, synthesizes numerous toxins, and incites explosive intraocular inflammation. The mechanisms involved in the rapid and explosive intraocular immune response have not been addressed. Because Toll-like receptors (TLRs) are integral to the initial recognition of organisms during infection, we hypothesized that the uniquely explosive immune response observed during B. cereus endophthalmitis is directly influenced by the presence of TLR2, a known gram-positive pathogen recognition receptor. To address this hypothesis, we compared the courses of experimental B. cereus endophthalmitis in wild type C57BL/6J mice to that of age-matched homozygous TLR2(-/-) mice. Output parameters included analysis of bacterial growth, inflammatory cell (PMN) infiltration, cytokine/chemokine kinetics, retinal function testing, and histology, with N≥4 eyes/assay/time point/mouse strain. B. cereus grew at similar rates to10(8) CFU/eye by 12 h, regardless of the mouse strain. Retinal function was preserved to a greater degree in infected TLR2(-/-) eyes compared to that of infected wild type eyes, but infected eyes of both mouse strains lost significant function. Retinal architecture was preserved in infected TLR2(-/-) eyes, with limited retinal and vitreal cellular infiltration compared to that of infected wild type eyes. Ocular myeloperoxidase activities corroborated these results. In general, TNFα, IFNγ, IL6, and KC were detected in greater concentrations in infected wild type eyes than in infected TLR2(-/-) eyes. The absence of TLR2 resulted in decreased intraocular proinflammatory cytokine/chemokine levels and altered recruitment of inflammatory cells into the eye, resulting in less intraocular inflammation and preservation of retinal architecture, and a slightly greater degree of retinal function. These results demonstrate TLR2 is an important component of the initial ocular response to B. cereus endophthalmitis.

Severe bacterial endophthalmitis: towards improving clinical outcomes.

Endophthalmitis is an infection and inflammation of the interior of the eye that can result in significant vision loss. This infection occurs as a result of the seeding of organisms into the interior of the eye following surgery (postoperative), trauma (post-traumatic) or an infection in another site in the body (endogenous). The general rate of endophthalmitis has remained steady over the past several years. However, the increased use of intraocular injections to treat various degenerative and inflammatory ocular diseases, in addition to the already large and growing number of invasive ocular surgeries, may increase the opportunities in which organisms can gain access to the eye. In most cases of endophthalmitis, useful vision can be retained if proper treatment is instituted. However, in severe cases of bacterial endophthalmitis, blindness often occurs despite treatment. This article summarizes information on endophthalmitis epidemiology, treatment issues and current regimens, and recent experimental and clinical efforts to improve the outcome of severe and blinding forms of bacterial endophthalmitis.

Rate of bacterial eradication by ophthalmic solutions of fourth-generation fluoroquinolones.

INTRODUCTION: Antibacterial activity of ophthalmic fourth-generation fluoroquinolones has traditionally been evaluated by comparing only their active ingredients, gatifloxacin and moxifloxacin. However, ophthalmic formulations of fourth-generation fluoroquinolones differ in terms of the inclusion of preservatives. While gatifloxacin ophthalmic solution 0.3% (Zymar; Allergan, Inc., Irvine, CA, USA) contains 0.005% benzalkonium chloride (BAK), moxifloxacin ophthalmic solution 0.5% (Vigamox; Alcon Laboratories, Inc., Fort Worth, TX, USA) is preservative-free. Recent studies have demonstrated that the presence of BAK dramatically affects the antibacterial activity of the ophthalmic formulation of gatifloxacin. This study was designed to compare the kill rates of ophthalmic solutions of fourth-generation fluoroquinolones against isolates of common ocular bacterial pathogens. METHODS: Approximately 5.6 log(10) colony-forming units (CFU)/mL of Haemophilus influenzae (n=1), Streptococcus pneumoniae (n=1), Staphylococcus aureus (n=2), methicillin-resistant Staphylococcus aureus (MRSA) (n=4), methicillinresistant Staphylococcus epidermidis (MRSE) (n=4), and fluoroquinolone-resistant S. epidermidis (n=1) were incubated with ophthalmic solutions of either gatifloxacin or moxifloxacin. Viable bacteria were quantified at specific time points up to 60 minutes. RESULTS: Gatifloxacin 0.3% completely eradicated H. influenzae and Strep. pneumoniae in 5 minutes, one of two S. aureus isolates in 15 minutes, and the other S. aureus isolate in 60 minutes. Gatifloxacin 0.3% completely killed all MRSA, MRSE, and fluoroquinolone-resistant S. epidermidis isolates in 15 minutes. Moxifloxacin 0.5% completely eradicated Strep. pneumoniae and one of four MRSA isolates in 60 minutes. All other isolates incubated with moxifloxacin 0.5% retained viable bacteria ranging from 1.8 to 4.4 log(10) CFU/mL. CONCLUSIONS: The ophthalmic solution of gatifloxacin 0.3% eradicated bacteria that frequently cause postoperative ocular infections substantially faster than did the ophthalmic solution of moxifloxacin 0.5%.

Bacillus cereus-induced permeability of the blood-ocular barrier during experimental endophthalmitis.

PURPOSE: The purpose of this study was to determine to what extent blood-retinal barrier (BRB) permeability occurred during experimental Bacillus cereus endophthalmitis and whether tight junction alterations were involved in permeability. METHODS: Mice were intravitreally injected with 100 colony-forming units of B. cereus, and eyes were analyzed at specific times after infection for permeability to fibrin and albumin, quantitation of intraocular plasma constituent leakage, production of inflammatory cytokines, and alterations in tight junction protein localization and expression at the level of the retinal pigment epithelium. RESULTS: B. cereus induced the leakage of albumin and fibrin into the aqueous and vitreous humor by 8 hours after infection. BRB permeability occurred as early as 4 hours and increased 13.30-fold compared with uninfected controls by 8 hours. Production of proinflammatory cytokines IL-6, MIP-1alpha, IL-1beta, and KC increased over the course of infection. In the retina, ZO-1 disruption began by 4 hours and was followed by decreasing occludin and ZO-1 expression at 4 and 8 hours, respectively. Tubulin condensation and RPE65 degradation occurred by 12 hours. A quorum-sensing mutant B. cereus strain caused BRB permeability comparable to that of wild-type B. cereus. Wild-type and mutant B. cereus sterile supernatants induced blood-ocular barrier permeability similarly to that of wild-type infection. CONCLUSIONS: These results indicate that BRB permeability occurs during the early stages of experimental B. cereus endophthalmitis, beginning as early as 4 hours after infection. Disruption of tight junctions at the level of the retinal pigment epithelium may contribute to barrier breakdown. Quorum-sensing dependent factors may not significantly contribute to BRB permeability.

Toward improving therapeutic regimens for Bacillus endophthalmitis.

PURPOSE: Bacillus cereus causes the most virulent and refractory form of endophthalmitis. The authors analyzed the effectiveness of early treatment with vancomycin or gatifloxacin, with or without dexamethasone, for experimental B. cereus endophthalmitis. METHODS: Rabbit eyes were injected intravitreally with 100 colony-forming units of B. cereus. At 2, 4, or 6 hours after infection, eyes were injected intravitreally with 0.1 mL gatifloxacin (0.3%), vancomycin (1.0%), either antibiotic plus dexamethasone, dexamethasone alone (1.0%), or PBS. Eyes were analyzed by electroretinography, bacterial quantitation, and antibiotic penetration analysis. Drug toxicity toward Müller cells, retinal pigment epithelium, and cones was also analyzed. RESULTS: Eyes treated at 2 hours with vancomycin or gatifloxacin, with or without dexamethasone, maintained higher ERG amplitudes than the dexamethasone alone and PBS control groups. Eyes treated with antibiotic plus dexamethasone at 6 hours had reduced retinal function compared to antibiotic treatment alone. With the exception of vancomycin with or without dexamethasone at 6 hours, all antibiotic treatments sterilized eyes. Only gatifloxacin reached aqueous concentrations greater than the minimal inhibitory concentration for B. cereus when measured at 8 hours. Neither gatifloxacin nor vancomycin was toxic to retinal cells in vitro. CONCLUSIONS: Early intravitreal injection of vancomycin or gatifloxacin improved the therapeutic outcome of B. cereus endophthalmitis. The addition of dexamethasone to antibiotic treatment did not provide a therapeutic benefit over antibiotics alone and appeared to reduce the antibiotic efficacy of vancomycin 6 hours after infection. In this model, delay in treatment past 6 hours significantly reduced the potential for salvaging useful vision.

Bacterial endophthalmitis: therapeutic challenges and host-pathogen interactions.

Endophthalmitis is an infection of the posterior segment of the eye that frequently results in loss of vision. This devastating result occurs despite prompt and often aggressive therapeutic and surgical intervention. Over the past decade, research has centered on determining the bacterial and host factors involved in this potentially blinding disease. The initial focus on the bacterial factors responsible for intraocular virulence has recently expanded into analysis the inflammatory response to infection, including the molecular and cellular interactions between the pathogen and host. This review discusses the epidemiology and therapeutic challenges posed by endophthalmitis, as well as recent findings from the analysis of interactions between the host and pathogen. Based on these findings, a model for the pathogenesis of endophthalmitis is presented. A more comprehensive understanding of the molecular and cellular interactions taking place between pathogen and host during endophthalmitis will expose possible therapeutic targets designed to arrest the infection and prevent vision loss.

Acute inflammation and loss of retinal architecture and function during experimental Bacillus endophthalmitis.

Rapid vision loss and explosive inflammation are devastating consequences of Bacillus endophthalmitis that have not been well defined. We therefore analyzed the evolution of intraocular inflammation and loss of retinal architecture and function during experimental Bacillus endophthalmitis. Mice were intravitreally injected with 100 CFU of B. cereus, and eyes were analyzed for bacterial growth, retinal function, architectural changes and retinal cellular stress, inflammatory cytokines, and infiltrating cells. Retinal electrophysiologic and structural changes began as early as 4 to 6 hr postinfection. Significant declines in retinal function paralleled the loss of retinal architecture. Glial fibrillary acidic protein (GFAP) was detected in retina, indicating potential stress. Polymorphonuclear leukocyte (PMN) infiltration into the vitreous began as early as 4 hr postinfection, coinciding with a significant increase in TNF-alpha in the eye. These results indicated that acute inflammation and detrimental architectural and electrophysiologic changes in the retina began earlier than once thought, suggesting that therapeutic intervention should be given at the earliest possible time to avoid vision loss during Bacillus endophthalmitis.

Role of swarming migration in the pathogenesis of bacillus endophthalmitis.

PURPOSE: Bacillus cereus causes one of the most rapidly blinding forms of bacterial endophthalmitis. Migration of B. cereus throughout the eye during endophthalmitis is a unique aspect of this disease that may contribute to intraocular virulence. This study was conducted to analyze the contribution of swarming and intraocular migration to the pathogenesis of experimental endophthalmitis. METHODS: Eyes were injected intravitreally with 100 colony-forming units (CFU) of either wild-type, nonswarming, or swarming-complemented strains of B. cereus. Pathogenicity was compared throughout the course of infection by biomicroscopy, histology, electroretinography, and bacterial and inflammatory cell quantitation. RESULTS: Wild-type, nonswarming, and swarming-complemented B. cereus strains grew to a similar number in the vitreous throughout the course of infection. Unlike the wild-type and swarming-complemented strains, the nonswarming mutant did not migrate to the anterior segment during infection. The rate of decrease in retinal responses of eyes infected with the all strains was similar, resulting in near complete elimination of retinal function by 12 hours. All Bacillus strains caused similar degrees of posterior segment inflammation and retinal destruction. However, the accumulation of inflammatory cells in the anterior chamber, hyphemae, and corneal ring abscesses did not occur in eyes infected with the nonswarming mutant. CONCLUSIONS: The deficiency in swarming had little effect on retinal function loss or the overall course or severity of experimental B. cereus endophthalmitis. However, a deficiency in swarming prevented Bacillus from migrating to the anterior segment, leading to less severe anterior segment disease.

Bacillus endophthalmitis: roles of bacterial toxins and motility during infection.

PURPOSE: Bacillus endophthalmitis is a highly explosive infection of the eye that commonly results in rapid inflammation and vision loss, if not loss of the eye itself, within a few days. Quorum-sensing-controlled toxins are essential to virulence during infection. Another unique characteristic of this disease is the ability of Bacillus to replicate rapidly and migrate to all parts of the eye. This study was conducted to determine the combined roles of toxins and motility during Bacillus endophthalmitis. METHODS: Rabbit eyes were injected intravitreally with approximately 100 cfu of wild type, nonmotile, or nonmotile/quorum-sensing-deficient Bacillus thuringiensis. Infection courses were analyzed by biomicroscopy, histology, electroretinography, and quantitation of bacteria and inflammatory cells. RESULTS: Infection with wild type B. thuringiensis resulted in complete retinal function loss by 18 hours after infection, whereas nonmotile B. thuringiensis infections required 30 hours to achieve a reduction of >90% in retinal function. Further attenuation of infection resulted from infection with the nonmotile/quorum-sensing-deficient B. thuringiensis strain, with approximately 90% retinal function loss occurring at 36 hours. Overall, the nonmotile and nonmotile/quorum-sensing-deficient mutants were significantly less virulent than wild-type B. thuringiensis. CONCLUSIONS: The results demonstrate that, in addition to quorum-sensing-controlled toxin production, bacterial migration within the eye contributed to the rapidly fulminant and destructive course of Bacillus endophthalmitis. Motility and quorum-sensing may therefore represent possible targets for the development of therapies designed to attenuate the devastating effects of Bacillus in the eye during endophthalmitis.